Subtractive single label comparative hybridization

ABSTRACT

Provided are methods of determining differences between nucleic acids in a test sample and a reference sample. In certain embodiments the methods are used for detecting and mapping chromosomal or genetic abnormalities associated with various diseases or with predisposition to various diseases, or to detecting the phenomena of large scale copy number variants. In particular, provided are advanced methods of performing array-based comparative hybridization that allow reproducibility between samples and enhanced sensitivity by using the same detectable label for both test sample and reference sample nucleic acids. Invention methods are useful for the detection or diagnosis of particular disease conditions such as cancer, and detecting predisposition to cancer based on detection of chromosomal or genetic abnormalities and gene expression level. Invention methods are also useful for the detection or diagnosis of hereditary genetic disorders or predisposition thereto, especially in prenatal samples. Moreover, invention methods are also useful for the detection or diagnosis of de novo genetic aberrations associated with post-natal developmental abnormalities.

FIELD OF THE INVENTION

The present invention relates to the detection and mapping ofchromosomal or genetic abnormalities, including those associated withvarious diseases or with predisposition to various diseases. In aparticular aspect, the present invention relates to the use of nucleicacids in comparative hybridization.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Comparative hybridization methods test the ability of two nucleic acidsto interact with a third target nucleic acid. In particular, comparativegenomic hybridization (CGH) is a method for detecting chromosomalabnormalities. CGH was originally developed to detect and identify thelocation of gain or loss of DNA sequences, such as deletions,duplications or amplifications commonly seen in tumors (Kallioniemi etal., Science 258:818-821, 1992). For example, genetic changes resultingin an abnormal number of one or more chromosomes (i.e., aneuploidy) haveprovided useful diagnostic indicators of human disease, specifically ascancer markers. Changes in chromosomal copy number are found in nearlyall major human tumor types. For a review, see Mittelman et al.,“Catalog of Chromosome Aberrations” in Cancer, Vol. 2 (Wiley-Liss,1994).

In addition, the presence of aneuploid cells has also been used as amarker for genetic chromosol al abnormalities. Various chromosomalabnormalities may occur in an estimated 0.5% of all live births. Forexample, Down's syndrome or trisomy 18 which has an incidence of about 1in 800 live births, is commonly the subject of a variety of prenatalscreens or diagnostic techniques. Chromosomal aneuploidies involvingchromosomes 13, 18, 21, X and Y account for up to 95% of all livebornchromosomal aberrations resulting in birth defects (Whiteman et al., Am.J. Hum. Genet. 49:A127-129, 1991), and up to 67% of all chromosomalabnormalities, including balanced translocations (Klinger et al., Am. J.Hum. Genet. 51:52-65, 1992).

CGH is useful to discover and map the location of genomic sequences withvariant copy number without prior knowledge of the sequences.Oligonucleotide probes directed to known mutations are not required forCGH. Early CGH techniques employ a competitive in situ hybridizationbetween test DNA and normal reference DNA, each labeled with a differentcolor, and a metaphase chromosomal spread. Chromosomal regions in thetest DNA, which are at increased or decreased copy number as compared tothe normal reference DNA can be quickly identified by detecting regionswhere the ratio of signal from the two different colors is altered. Forexample, those genomic regions that have been decreased in copy numberin the test cells will show relatively lower signal from the test DNAthan the reference (compared to other regions of the genome (e.g., adeletion)); while regions that have been increased in copy number in thetest cells will show relatively higher signal from the test DNA (e.g., aduplication). Where a decrease or an increase in copy number is limitedto the loss or gain of one copy of a sequence, CGH resolution is usuallyabout 5-10 Megabases (Mb).

CGH has more recently been adapted to analyze individual genomic nucleicacid sequences rather than a metaphase chromosomal spread. Individualnucleic acid sequences are arrayed on a solid support, and the sequencescan represent the entirety of one or more chromosomes, or the entiregenome. The hybridization of the labeled nucleic acids to the arraytargets is detected using different labels, e.g., two colorfluorescence. Thus, array-based CGH with a plurality of individualnucleic acid sequences allows one to gain more specific information thana chromosomal spread, is potentially more sensitive, and facilitates theanalysis of samples.

For example, in a typical array-based CGH, equal amounts of totalgenomic nucleic acid from cells of a test sample and a normal referencesample are labeled with two different colors of fluorescent dye andco-hybridized to an array of BACs, which contain the cloned nucleic acidfragments that collectively cover the cell's genome. The resultingco-hybridization produces a fluorescently labeled array, the colorationof which reflects the competitive hybridization of sequences in the testand reference genomic DNAs to the homologous sequences within thearrayed BACs. Theoretically, the copy number ratio of homologoussequences in the test and reference genomic nucleic acid samples shouldbe directly proportional to the ratio of their respective coloredfluorescent signal intensities at discrete BACs within the array.Array-based CGH is described in U.S. Pat. Nos. 5,830,645 and 6,562,565for example, using target nucleic acids immobilized on a solid supportin lieu of a metaphase chromosomal spread.

When combining more than one color or type of labeled nucleic acid in ahybridization mixture, the relative concentrations and/or labelingdensities may be adjusted for various purposes. Adjustments may be madeby selecting appropriate detection reagents (avidin, antibodies and thelike), or by the design of the microscope filters among otherparameters. When using quantitative image analysis, mathematicalnormalization can be used to compensate for general differences in thestaining intensities of different colors. Thus, the use of differentlabels to distinguish test from reference genomic nucleic acids intraditional CGH entails additional refinements or adjustments thatcomplicate sample processing, standardization across samples, andevaluation of the results obtained. For example, when using visualobservation or photography of the results, the individual colorintensities need to be adjusted for optimum observability of changes intheir relative intensities.

U.S. Patent Application Publication Number 2005/0260665, (hereinafter“the '665 application”) which is hereby incorporated by reference hereinin its entirety including all figures and tables, discloses single-labelCGH methods.

One approach of the single label CGH methods disclosed in the '665application is referred to as an “additive” approach. In this approach,the test sample nucleic acids comprise a first tag; and the referencesample nucleic acids comprise a second tag. Following hybridization, thesurface is contacted with a first complex containing a detectable labeland a first entity, such that the first complex selectively binds withthe first tag. The next step comprises determining the location andamount of the detectable label bound to the array surface (i.e., to“read” the array). Once the array is read to determine the amount ofdetectable label associated with nucleic acid that comprises the firsttag, the surface is then contacted with a second complex containing thesame detectable label as present in the first complex and containing asecond entity, such that the second complex selectively binds with thesecond tag. The array is then read a second time to determine thelocation and amount of the total detectable label representing bothnucleic acids hybridized to the surface. The last step comprises usingthe results of the two reads to determine the amount of the hybridizednucleic acid that is associated with the second tag.

A second approach of the single label CGH methods disclosed in the '665application is referred to as an “subtractive” approach. In the“subtractive” approach, the linkage used to attach the detectable labelto the test nucleic acid and the reference nucleic acid is different,allowing for selective cleavage or removal of one linkage over that ofthe other. As a first step, the total detectable signal on the array,which represents label linked to both the test sample and the referencesample nucleic acids hybridized to the array, is first positionallyquantified. The array is then subjected to a condition or treatment thatcauses selective cleavage of the linker such that the label is strippedfrom either the hybridized test or reference nucleic acids, whicheverhas the susceptible linkage. The remaining signal representing nucleicacid that is not linked to the susceptible linker is then positionallyquantified. The next step includes using the results of the two reads todetermine the amount of the hybridized nucleic acid that is attached tothe label via the susceptible linkage. In a preferred approach, thesignal representing the nucleic acid that is linked to the label by thesusceptible linker is determined by subtracting the remaining signalfollowing selective removal from the total signal. The signal from thetwo samples thus determined can be used to identify differences betweenthe test sample genomic nucleic acids and the reference sample genomicnucleic acids so as to detect chromosomal or genetic abnormalitiesassociated with the test sample nucleic acid.

As described below, improvements in comparative hybridization methodsincluding CGH are provided. In particular, provided are improved methodsthat are variations of the “subtractive” methods disclosed in the '665application.

SUMMARY OF THE INVENTION

The present invention overcomes the limitations of using differentlabels in comparative hybridization (for example, different fluorescentcolors that must be read at two different wavelengths) and inparticular, comparative genomic hybridization methods. Accordingly,provided is a method of performing comparative hybridization bycomparing the amount of test and reference nucleic acids hybridized to anucleic acid array, the amounts determined by detecting a signal fromthe hybridized nucleic acids which are labeled with the same detectablelabel. This method is applicable to comparative hybridization methods ingeneral and to CGH in particular. Accordingly, reference to CGH wherethe test and reference nucleic acid is genomic nucleic acid should beunderstood to encompass methods where the test and reference nucleicacids are other than genomic nucleic acids. By the same token, it willbe understood that the type of label used is not critical and thatvarious labels described herein and known in the art and yet to bediscovered may be used in this invention and that reference to a singletype label (e.g. fluorescent label) in any of the embodiments of theinvention disclosed herein should be understood to include such othertypes of labels.

In a preferred embodiment of the methods provided herein, CGH isperformed using two samples of genomic nucleic acids; a test samplecontaining genomic nucleic acids, and a reference or control samplecontaining genomic nucleic acids with no known chromosomal or geneticabnormalities. The test sample and the reference sample areco-hybridized to a nucleic acid array that contains a plurality ofnucleic acids or nucleic acid segments spotted onto a surface (such as aglass slide) at discrete locations. The array may contain target nucleicacid markers for certain known genetic mutations or disease states, ormay represent (in aggregate) an entire chromosome, or the fullchromosomal complement to obtain a genetic profile similar tokaryotyping. In these approaches the detectable label may be attached tothe test and reference nucleic acids before hybridization or afterhybridization. In another approach, the detectable label may be attachedto one of the test or reference nucleic acids before hybridization whilethe label is attached to the other of the test or reference nucleic acidafter hybridization. The detectable label may be attached covalently ornon-covalently such as by a ligand-receptor interaction or byhybridization between complementary nucleotide sequences.

In some embodiments of the methods provided herein, the test andreference samples are labeled with a detectable label; preferably thetest and reference samples are labeled with the same detectable label;preferably the detectable label is a fluorochrome; preferably thedetectable label is dCTP-Cy3. In certain aspects, methods are providedthat allow for the use of a single label to determine the relativeamount of test and reference nucleic acids hybridized to the array. Thismay be achieved by various approaches as disclosed herein.

In a variation of the “subtractive approach”, the test and referencenucleic acids are labeled with the same detectable label, andco-hybridized to an array. As a first step, the total detectable signalon the array, which represents label linked to both the test sample andthe reference sample nucleic acids hybridized to the array, is firstpositionally quantified. The array is then subjected to a condition ortreatment that causes selective degradation and/or selective removal ofeither the hybridized test nucleic acid or the reference nucleic acid.The remaining signal representing nucleic acid that is not selectivelyremoved or degraded is then positionally quantified. The next stepincludes using the results of the two reads to determine the amount ofthe hybridized nucleic acid that is subject to being selectivelyremoved. In a preferred approach, the signal representing the nucleicacid that is linked to the label by the susceptible linker is determinedby subtracting the remaining signal following selective removal from thetotal signal. The signal from the two samples thus determined can beused to identify differences between the test sample genomic nucleicacids and the reference sample genomic nucleic acids so as to detectchromosomal or genetic abnormalities associated with the test samplenucleic acid.

In one aspect, a method of determining differences between nucleic acidin a test sample and a reference sample is provided. The method involvesamplifying nucleic acid sequence from the test sample nucleic acid andamplifying nucleic acid sequence from the reference sample nucleic acid,where one of the amplification reactions is conducted using dUTP and notdTTP and the other is conducted using dTTP and not dUTP; hybridizing toa nucleic acid array a solution comprising the amplified test sample andamplified reference sample; and determining the relative amount ofhybridized test and reference nucleic acids bound to the array. Incertain embodiments of the methods provided herein, determining therelative amount of hybridized test and reference nucleic acids includesa) determining a signal for the detectable label hybridized to the arrayrepresenting the total of hybridized test and reference nucleic acid; b)treating the hybridized nucleic acids with an enzyme that selectivelydegrades DNA having uracil residues; and c) determining a signal for thedetectable label hybridized to the array following step b), which signalrepresents one of the hybridized test or reference nucleic acid.

In particularly preferred embodiments of the methods provided herein,the enzyme that selectively degrades DNA having uracil residues isuracil-DNA N-glycosylase (UNG).

In another aspect, a method of determining differences between nucleicacid in a test sample and a reference sample is provided, where themethod involves: (a) contacting under hybridization conditions a testsample containing nucleic acids and a reference sample containingnucleic acids to a surface containing a plurality of nucleic acidsegments each immobilized at discrete locations on the surface, wherethe test sample and the reference sample are labeled before or afterhybridization with the same detectable label; (b) determining thelocation and amount of the detectable label linked to nucleic acidshybridized to the surface; (c) selectively removing either thehybridized test sample nucleic acids or the hybridized reference samplenucleic acids; (d) determining the location and amount of the detectablelabel linked to nucleic acids hybridized to the surface following step(c); and (e) comparing the results of step (b) to the results of step(d) to detect differences in the nucleic acids of the test sample andreference sample.

In some preferred embodiments of the methods provided herein, the stepof selectively removing hybridized test nucleic acids or referencenucleic acids is performed by subjecting the nucleic acids to an enzymethat selectively degrades DNA having certain properties; preferably anenzyme that degrades DNA having uracil residues; more preferably theenzyme that selectively degrades DNA having uracil residues isuracil-DNA N-glycosylase (UNG). In some embodiments of the methodsprovided herein, the step of selectively removing hybridized testnucleic acids or reference nucleic acids by subjecting nucleic acids toan enzyme that selectively degrades DNA having uracil residues isachieved by (1) amplifying sequence from a test sample and amplifyingsequence from a reference sample nucleic acid, where one of theamplification reactions is conducted using dUTP and not dTTP and theother is conducted using dTTP and not dUTP; (2) hybridizing theamplified nucleic acids; and (3) treating the hybridized nucleic acidswith an enzyme that selectively degrades DNA having uracil residues.

The methods provided herein may be used to detect any differencesbetween nucleic acids in a test sample and a reference sample, includingdifferences in the amount of nucleic acids having a particular sequenceor differences in nucleic acid sequences. In particularly preferredembodiments, the methods are used to detect genetic abnormalities in thetest sample. The methods provided herein may by applied to CGH using achromosomal spread or array-based CGH. In some preferred embodiments,the methods provided may be used to compare the expression of genes in atest sample versus that of a reference sample.

In one aspect of the methods provided herein, a method of performingcomparative hybridization is provided. The method includes comparing theamount of test and reference nucleic acids hybridized to a nucleic acidarray, wherein the amount of hybridized test and reference nucleic acidsis determined by detecting a signal from the hybridized nucleic acidswhich are labeled with the same detectable label. In one embodiment, theamount of hybridized test and reference nucleic acids are determined by:a) determining a signal for the detectable label hybridized to the arrayrepresenting the total of hybridized test and reference nucleic acid; b)treating the hybridized nucleic acids to selectively remove one of thetest or reference nucleic acids; c) determining a signal for thedetectable label hybridized to the array following step b), whichrepresents one of the hybridized test or reference nucleic acid; and d)determining a signal for the other of the hybridized test or referenceby using the signal from c) and b).

In certain preferred embodiments of the methods provided herein, thestep of amplifying sequence from a test sample and amplifying sequencefrom a reference sample involves amplifying genomic DNA in the samplesis conducted using random priming such as is well known in the art.Alternatively, the step of amplifying sequence from a test sample andamplifying sequence from a reference sample may involve using RNA togenerate cDNA and amplifying the cDNA using random priming and oramplifying specific sequences using particular primers. In certainpreferred embodiments, the amplification reaction may be performed usingone or more labeled nucleotides as a means to label the amplifiednucleic acids with a detectable label; preferably both test andreference sample nucleic acids are amplified with the same labelednucleotide; preferably the labeled nucleotide is dCTP-Cy3.

In another aspect, a method of comparing the expression of genes in atest sample versus that of a reference sample is provided. The methodincludes comparing the amount of cDNA prepared from mRNA of a testsample and cDNA prepared from mRNA of a reference sample hybridized to anucleic acid array, the amount of hybridized test and reference cDNAdetermined by detecting a signal from the hybridized cDNA which islabeled with the same detectable label. The method involves amplifyingnucleic acid sequence from cDNA prepared from RNA of the test sample andamplifying nucleic acid sequence from cDNA prepared from RNA of thereference sample, where one of the amplification reactions is conductedusing dUTP and not dTTP and the other is conducted using dTTP and notdUTP; hybridizing to the nucleic acid array a solution comprising theamplified test sample and amplified reference sample; and determiningthe relative amount of hybridized test and reference nucleic acids boundto the array. In certain embodiments of the methods provided herein,determining the relative amount of hybridized test and reference nucleicacids includes a) determining a signal for the detectable labelhybridized to the array representing the total of hybridized test andreference nucleic acid; b) treating the hybridized nucleic acids with anenzyme that selectively degrades DNA having uracil residues; and c)determining a signal for the detectable label hybridized to the arrayfollowing step b), which signal represents one of the hybridized test orreference nucleic acid.

U.S. Patent Application Publication Number 2007/0122820, herebyincorporated by reference in its entirety, discloses CGH methods todetect genetic abnormalities including balanced translocations usingprobes to detect specific sequences. In one aspect, a method is providedin which the methods involving probes disclosed in the 2007/0122820application are used in conjunction with the methods provided herein.

The term “tag” as used herein, refers to any physical molecule directlyor indirectly associated with a nucleic acids of a sample such thatsubstantially all individual nucleic acid segments of that sample can bemarked, purified, or captured via the same tag. The tag may be a memberof a specific binding pair such as a ligand-receptor or a pair ofoligonucleotides with a complementary sequence. The tag entityinteraction referred to herein is understood to be a specific bindingpair such as a ligand/receptor binding pair or a pair oligonucleotideswith a complementary sequence. A tag/entity combination should be chosenso that the it does not appreciably interact with the other tag entitycombination that is used together in comparative hybridization. Thisallows one to identify hybridized test from the reference nucleic acidby the specific interaction associated with each tag/entity.

In a preferred embodiment, the tag includes a unique oligonucleotide“capture sequence,” which refers to a sequence of nucleotides that isessentially unique to the assay. In this case, the reactive entityincludes an oligonucleotide complementary to the unique oligonucleotidecapture sequence associated with one sample of nucleic acid segments.Preferably, the reactive entity complementary oligonucleotide is in adendrimeric construct to provide a multiplicity of the detectable label.

As used herein, specific binding pair members include antigen-antibody,biotin-avidin, hormone-receptor, receptor-ligand, enzyme-substrate,IgG-protein A, and the like.

As used herein, “nucleic acid” refers to segments or portions of DNA,cDNA, and/or RNA. Nucleic acid may be derived or obtained from anoriginally isolated nucleic acid sample from any source (e.g., isolatedfrom, purified from, amplified from, cloned from, reverse transcribedfrom sample DNA or RNA) or may be synthesized de novo. Nucleic acidincludes an oligonucleotide, nucleotide or polynucleotide, and fragmentsor portions thereof, and to DNA or RNA of genomic or synthetic originthat may be single or double stranded, and represent the sense orantisense strand.

“Genomic nucleic acid” refers to some or all of the DNA from the nucleusof a cell. In some embodiments, genomic DNA may include sequence fromall or a portion of a single gene or from multiple genes, sequence fromone or more chromosomes, or sequence from all chromosomes of a cell. Incontrast, the term “total genomic nucleic acid” is used herein to referto the full complement of DNA contained in the genome of a cell. As iswell known, genomic nucleic acid includes gene coding regions, introns,5′ and 3′ untranslated regions, 5′ and 3′ flanking DNA and structuralsegments such as telomeric and centromeric DNA, replication origins, andintergenic DNA. Genomic nucleic acid may be obtained from the nucleus ofa cell, or recombinantly produced. Genomic DNA also may be transcribedfrom DNA or RNA isolated directly from a cell nucleus. PCR amplificationalso may be used. Methods of purifying DNA and/or RNA from a variety ofsamples are well-known in the art.

As used herein, “cDNA” refers to DNA which is copied from RNA. cDNAcopied from mRNA does not include the various non-coding sequencescharacteristic of genomic DNA.

As used herein, “chromosomal abnormality” refers to any difference inthe DNA sequence from a wild-type or normal cell. A chromosomalabnormality may reflect a difference between the full genetic complementof all chromosomes contained in an organism, or any portion thereof, ascompared to a normal full genetic complement of all chromosome in thatorganism. For example, a chromosomal abnormality may include a change inchromosomal copy number (e.g. aneuploidy), or a portion thereof (e.g.deletions, amplifications); or a change in chromosomal structure (e.g.,translocations, mutations). “Aneuploid cell” or “aneuploidy” as usedherein, refers to a cell having an abnormal number of at least onechromosome in interphase. For example, a normal human cell contains atotal of 46 chromosomes in interphase, or 2 copies of each ofchromosomes 1 through 22, and 2 sex chromosomes (XX or XY). An abnormalchromosomal copy number is any number other than two of the normalchromosomal complement of two copies of chromosomes 1 through 22, andany combination other than two of the sex chromosomes X and Y.

As used herein, “genetic abnormality” refers to a chromosomalabnormality that is known to be associated with a particular diseasecondition (e.g., a specific gene mutation causing a dysfunctionalprotein directly causing a disease state). A chromosomal or geneticabnormality may be hereditary, i.e., passed from generation togeneration.

A “sample” as used herein may be acquired from essentially any diseasedor healthy organism, including humans, animals and plants, as well ascell cultures, recombinant cells, cell components and environmentalsources. Samples may be from any animal, including by way of example andnot limitation, humans, dogs, cats, sheep, cattle, and pigs. Samples canbe a biological tissue, fluid or specimen. Samples may include, but arenot limited to, amniotic fluid, blood, blood cells, cerebrospinal fluid,fine needle biopsy samples, peritoneal fluid, plasma, pleural fluid,saliva, semen, serum, sputum, tissue or tissue homogenates, tissueculture media, urine, and the like. Samples may also be processed, suchas sectioning of tissues, fractionation, purification, or cellularorganelle separation.

A “test sample” comprises genomic nucleic acids typically from a patientor a cell population suspected of, or being screened for, nucleic acidcontaining a chromosomal or genetic abnormality. A “reference sample”comprises genomic nucleic acids typically from a normal individual orwild-type cell population with a normal genetic profile. A “test sample”or “reference sample” also may comprise mRNA from which cDNA can bemade.

The genomic nucleic acids from the test and reference samples arecontacted under hybridization conditions to a surface containing aplurality of nucleic acid segments, each immobilized at discretelocations on the surface. The term “hybridization” as used herein,refers to the pairing of substantially complementary nucleotidesequences (strands of nucleic acid) to form a duplex or heteroduplexthrough formation of hydrogen bonds between complementary base pairs. Itis a specific, i.e., non-random, interaction between two complementarypolynucleotides. Hybridization and the strength of hybridization (i.e.,the strength of the association between the nucleic acids) is influencedby such factors as the degree of complementary between the nucleicacids, stringency of the conditions involved, and the T_(m) of theformed hybrid.

Genomic nucleic acids of the test sample may be linked to a detectablelabel via a first linkage. Genomic nucleic acids of the reference samplemay be linked to the same detectable label via a second linkage.

A “detectable label” as used herein refers any moiety that generates adetectable signal by spectroscopic, photochemical, biochemical,immunochemical, electromagnetic, radiochemical, or chemical means, suchas fluorescence, chemifluoresence, or chemiluminescence, or any otherappropriate means. Preferred detectable labels include fluorescent dyemolecules, or fluorophores, such as fluorescein, phycoerythrin, Cy3™,Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, FAM,JOE, TAMRA, tandem conjugates such as phycoerythrin-Cy5™, and the like.Cy3™, and Cy5™ are commonly used together in two color detectionsystems. In single label CGH, Cy3™ is preferred over Cy5™. Thedetectable label may be linked by covalent or non-covalent means tonucleic acids. Alternatively, a detectable label may be linked such asby directly labeling a molecule that achieves binding to another nucleicacid via a ligand-receptor binding pair arrangement or other suchspecific recognition molecules.

A “linkage” of a detectable label as used herein, means that the labelis physically associated with genomic nucleic acids in a sample. In oneembodiment, either the first linkage or the second linkage issusceptible to selective removal. i.e., the linkage of one label but notthe other is susceptible to cleavage or separation allowing the label tobe separated from the nucleic acid. Examples of pairs of linkages (i.e.,a differential linkage where one linker of the pair is susceptible toselective removal) include linkage via two different chemical linkers,two different oligonucleotides, or two different peptide sequences,wherein the chemical linkers, oligonucleotides or peptide sequencesdiffer in susceptibility to temperature, pH hydrolysis, radiation (e.g.,nucleotide stretches or chemical entities sensitive to ultravioletradiation; e.g., photocleavable entities), oxidative conditions,atmospheric conditions (e.g., exposure to ozone), buffer conditions,hydrolysis by an external agent (e.g., an enzyme, such as a restrictionendonuclease or a homing endonuclease), or chemical cleavage (e.g.,linkers containing a diol that can be selectively cleaved usingsaturated aqueous NaIO₄ for 30-40 minutes, or linkers containing adisulfide that can be cleaved with dithiothreitol or any otherappropriate reducing reagent, such as those available from FidelitySystems, Inc. Gaithersburg, Md.).

By “susceptibility” is meant that the detectable label associated withthe nucleic acids containing the susceptible linkage is physicallydissociated from about 80% or more, preferably 90% or more, morepreferably 95% or more of the member nucleic acids of that sample.Treatments that remove less than 95% can be tolerated especially whenthe deficiency in removal can be calculated and factored into the finalresults. Other differential linkages include different chemical couplingor physical interactions of the label with nucleic acids of either thetest or reference sample in the labeling process. The nucleic acids ofthe sample(s) may be labeled before hybridization to the array, or afterhybridization to the array. In all of these examples, a susceptiblelinkage is created to render the nucleic acids of one sample subject toselective removal of the label associated with the nucleic acids in thatsample, following an initial read of the hybridization. Thus, the samelabel may be read at two time points: (i) to detect the signal from thedetectable label of both the test and reference nucleic acids hybridizedto the array; and (ii) to detect the signal from the nucleic acidshybridized to the array that do have the label attached by a linker thatis susceptible to the treatment. Subtraction of these two readingsyields a value representing the hybridized nucleic acid that is labeledvia the linkage that was resistant to the removal treatment.

In a one approach, the label may be non-covalently associated with thesample or reference nucleic acid, thus allowing the nucleic acids tofirst be hybridized to the target before the label is attached. In thiscase, a first unique oligonucleotide can be attached to either the testor reference sample nucleic acids, wherein this oligonucleotide containsa unique hybridization sequence and a recognition site for a restrictionendonuclease, a homing endonuclease, or a rare-cutting endonuclease asis known in the art and commercially available, for example fromFermentas Life Sciences (e.g., I-SceI). The other of the nucleic acidsis preferably linked to a second oligonucleotide that contains adifferent unique hybridization sequence but does not contain thisrecognition site. Both nucleic acids with the attached oligonucleotidesare separately detected using another detectably labeled oligonucleotidewhich is complementary to one or the other hybridization sequence. Suchdetectably labeled complementary oligonucleotide may be a dendrimericcomplex. Using this embodiment, following application of both labeledoligonucleotides to the hybridized array, label associated via the firstunique oligonucleotide may then be selectively removed by contact withthe endonuclease specific for the recognition site in the first uniqueoligonucleotide.

A “dendrimer” as used herein, is an artificially manufactured orsynthesized polymeric molecule built up from branched units calledmonomers. In a preferred embodiment, the monomers are DNA moleculeswhich associate by base pairing to assemble (see, e.g., U.S. Pat. Nos.5,175,270; 5,484,904; and 5,487,973). Other monomers include, but arenot limited to, primary amines (see, e.g., U.S. Pat. No. 5,530,092);polyamidoamines, polyethyleneimines, and polypropyleneimines (see, e.g.,U.S. Pat. Nos. 5,393,797; 5,393,795; 5,560,929; and 5,387,617);peptides; and other nucleic acids. Various tags (e.g., anoligonucleotide) may be attached to a terminal end of a dendrimerpolymer or may be incorporated into the internal structure of thedendrimer. Attachment includes covalent attachment (e.g., the 3′ end ofan oligonucleotide is covalently attached to a terminal end of adendrimer branch) as well as non-covalent interactions (e.g., nucleicacid hybridization). By incorporating a multiplicity of labels into thedendrimer, hybridization signal intensity is dramatically enhanced.

According to yet a further aspect of the invention, there is provided amethod of comparing the expression of genes in a test sample versus thatof reference sample. The first step of the method includes contactingunder hybridization conditions cDNA prepared from mRNA of a test sampleand cDNA prepared from mRNA of a reference sample to a surfacecontaining a plurality of nucleic acid segments each immobilized atdiscrete locations on the surface. In this case, the test sample cDNAand the reference sample cDNA are labeled before or after hybridizationwith the same detectable label which is linked to the cDNA of the testsample via a first linkage, and to the cDNA of the reference sample viaa second linkage. Either the first linkage or the second linkage issusceptible to selective removal and the detectable label linked tonucleic acids hybridized to the surface determined. The location andamount of detectable label linked to nucleic acids hybridized thesurface of the support is determined. The label is then selectivelyremoved from either the hybridized test sample cDNA or the hybridizedreference sample cDNA. The location and amount of the detectable labelremaining on the support is then determined and represents one of thesamples. The difference between the location and amount remaining afterremoval compared and the location and amount prior to removal representsthe other of the samples. The relative amount of each sample nucleicacid hybridized to the array reflects the expression of genes in thetest sample compared to the reference sample.

The term “expression array” refers to a collection of cDNA sequencesrepresenting the complement of mRNA present in a cell at a particulartime. A cDNA expression array may be prepared by oligo dT priming orrandom priming with hexomers. The random primer oligos may have 5′ligatable ends. After priming and extension to produce cDNA, theligatable ends of the cDNA are ligated to a capture sequence via abridging oligo. cDNA with capture sequences may then be hybridized to asolid phase containing immobilized nucleic acid probe sequences. Thecapture sequence may be detected with an appropriate labeled reagent(e.g. labeled dendrimeric nucleic acid) with a single strandedsegment(s) (a.k.a. tail(s)) for the capture sequence. Exemplary suppliesand protocols for preparing an expression array are available frommanufacturers, for example, Genisphere, Inc.

Any of the embodiments disclosed herein for comparative genomichybridization may be used to detect a chromosomal abnormality or geneticabnormality in any patient including adults, children and neonates.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided improvedmethods of performing single label array-based comparative genomichybridization (CGH) to detect a chromosomal abnormality in a testsample, or to diagnose a genetic abnormality in an individual. Inparticular, variations of the subtractive methods described in the '665application are provided. CGH is a molecular cytogenetics approach thatcan be used to detect regions in a genome undergoing quantitativechanges, e.g., gains or losses of sequence or copy numbers. CGH isespecially useful in the analysis and diagnosis of cancer, and theanalysis and diagnosis of genetic disorders, such as in prenataldiagnosis. CGH reactions are typically used to compare the geneticcomposition of an unknown test sample with a known normal referencesample.

Sources of Genomic Nucleic Acids

In one aspect, the methods of the present invention can be used todetect a chromosomal abnormality in a test sample. In a preferredembodiment, the test sample is obtained from a patient. In anotherpreferred embodiment, the test sample contains cells, tissues or fluidobtained from a patient suspected of having a pathology or a conditionassociated with a chromosomal or genetic abnormality. The causality,diagnosis or prognosis of the pathology or condition may be associatedwith genetic defects, e.g., with genomic nucleic acid basesubstitutions, amplifications, deletions and/or translocations. The testsample may be suspected of containing cancerous cells or nucleic fromsuch cells. Samples may include, but are not limited to, amniotic fluid,biopsies, blood, blood cells, bone marrow, cerebrospinal fluid, fecalsamples, fine needle biopsy samples, peritoneal fluid, plasma, pleuralfluid, saliva, semen, serum, sputum, tears, tissue or tissuehomogenates, tissue culture media, urine, and the like. Samples may alsobe processed, such as sectioning of tissues, fractionation,purification, or cellular organelle separation.

Methods of isolating cell, tissue or fluid samples are well known tothose of skill in the art and include, but are not limited to,aspirations, tissue sections, drawing of blood or other fluids, surgicalor needle biopsies, and the like. Samples derived from a patient mayinclude frozen sections or paraffin sections taken for histologicalpurposes. The sample can also be derived from supernatants (of cellcultures), lysates of cells, cells from tissue culture in which it maybe desirable to detect levels of mosaicisms, including chromosomalabnormalities and copy numbers.

In a preferred embodiment, a sample suspected of containing cancerouscells is obtained from a human patient. Samples can be derived frompatients using well-known techniques such as venipuncture, lumbarpuncture, fluid sample such as saliva or urine, tissue or needle biopsy,and the like. In a patient suspected of having a tumor containingcancerous cells, a sample may include a biopsy or surgical specimen ofthe tumor, including for example, a tumor biopsy, a fine needleaspirate, or a section from a resected tumor. A lavage specimen may beprepared from any region of interest with a saline wash, for example,cervix, bronchi, bladder, etc. A patient sample may also include exhaledair samples as taken with a breathalyzer or from a cough or sneeze. Abiological sample may also be obtained from a cell or blood bank wheretissue and/or blood are stored, or from an in vitro source, such as aculture of cells. Techniques for establishing a culture of cells for useas a sample source are well known to those of skill in the art.

In another aspect, the methods of the present invention can be used todetect a chromosomal or genetic abnormality in a fetus. Prenataldiagnosis of a fetus may be indicated for women at increased risk ofcarrying a fetus with chromosomal or genetic abnormalities. Risk factorsare well known in the art, and include, for example, advanced maternalage, abnormal maternal serum markers in prenatal screening, chromosomalabnormalities in a previous child, a previous child with physicalanomalies and unknown chromosomal status, parental chromosomalabnormality, and recurrent spontaneous abortions.

The invention methods can be used to perform prenatal diagnosis usingany type of embryonic or fetal cell. Fetal cells can be obtained throughthe pregnant female, or from a sample of an embryo. Thus, fetal cellsare present in amniotic fluid obtained by amniocentesis, chorionic villiaspirated by syringe, percutaneous umbilical blood, a fetal skin biopsy,a blastomere from a four-cell to eight-cell stage embryo(pre-implantation), or a trophectoderm sample from a blastocyst(pre-implantation or by uterine lavage). Body fluids with sufficientamounts of genomic nucleic acid also may be used.

The method of the present invention utilizes a first population ofgenomic nucleic acids obtained from the test sample, and a secondpopulation of genomic nucleic acids obtained from a reference sample.The reference sample may be any cells, tissues or fluid as providedherein, obtained from an individual, or any cell culture or tissueculture, that does not contain any genetic abnormality, i.e., that has anormal genetic complement of all chromosomes.

Association of Label with Genomic Nucleic Acids

The genomic nucleic acids of both the test sample and the referencesample are associated with the same detectable label, either prior to orsubsequent to hybridization. In preferred embodiments, the label isdetectable by optical means, and is most preferably a fluorescent labelor fluorophore. The detectable label can be incorporated into,associated with or conjugated to a nucleic acid. The association betweenthe nucleic acid and the detectable label can be covalent ornon-covalent. According to the methods of the present invention, thesame detectable label is used to label both the genomic nucleic acids ofthe test sample and the genomic nucleic acids of the reference sample.Label can be attached by spacer arms of various lengths to reducepotential steric hindrance or impact on other useful or desiredproperties. See, e.g., Mansfield, Mol. Cell. Probes 9:145-156, 1995.

Useful labels include, e.g., fluorescent dyes (e.g., Cy5™, Cy3™, FITC,rhodamine, lanthamide phosphors, Texas red), ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I,¹³¹I, electron-dense reagents (e.g., gold), enzymes, e.g., as commonlyused in an ELISA (e.g., horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), calorimetric labels (e.g., colloidalgold), magnetic labels (e.g., Dynabeads™), biotin, dioxigenin, orhaptens and proteins for which antisera or monoclonal antibodies areavailable. The label can be directly incorporated into the nucleic acidto be detected, or it can be attached to a probe (e.g., anoligonucleotide) or antibody that hybridizes or binds to the nucleicacid to be detected.

In preferred embodiments, the detectable label is a fluorophore. Theterm “fluorophore” as used herein refers to a molecule that absorbs aquantum of electromagnetic radiation at one wavelength, and emits one ormore photons at a different, typically longer, wavelength in response.Suitable fluorescent moieties include the following fluorophores knownin the art:

4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives (acridine and acridine isothiocyanate), Alexa Fluor® 350,Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor™ 568,Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes),5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS) N-(4-anilino-1-naphthyl)maleimide, anthranilamide, Black HoleQuencher™ (BHQ™) dyes (Biosearch Technologies), BODIPY® R-6G, BOPIPY®530/550, BODIPY® FL, Brilliant Yellow, coumarin and derivatives(coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151)), Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®, cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,diethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), Eclipse™(Epoch Biosciences Inc.), eosin and derivatives (eosin and cosinisothiocyanate), erythrosin and derivatives (erythrosin B and erythrosinisothiocyanate), ethidium, fluorescein and derivatives

-   -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)    -   2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate (FITC)    -   hexachloro-6-carboxyfluorescein (HEX)    -   QFITC (XRITC)    -   tetrachlorofluorescein (TET)        fluorescamine, IR144, IR1446, Malachite Green isothiocyanate,        4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,        pararosaniline, Phenol Red, B-phycoerythrin, R-phycoerythrin,        o-phthaldialdehyde, Oregon Green®, propidium iodide, pyrene and        derivatives (pyrene, pyrene butyrate, succinimidyl 1-pyrene        butyrate), QSY® 7, QSY® 9, QSY® 21, QSY®35 (Molecular Probes),        Reactive Red 4 (Cibacron® Brilliant Red 3B-A), rhodamine and        derivatives:    -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride    -   rhodamine (Rhod)    -   rhodamine B    -   rhodamine 123    -   rhodamine green    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)        N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl        rhodamine, tetramethyl rhodamine isothiocyanate (TRITC),        riboflavin, rosolic acid, and terbium chelate derivatives.

Other fluorescent nucleotide analogs can be used, see, e.g., Jameson,Meth. Enzymol. 278:363-390, 1997; Zhu, Nucl. Acids Res. 22:3418-3422,1994. U.S. Pat. Nos. 5,652,099 and 6,268,132 also describe nucleosideanalogs for incorporation into nucleic acids, e.g., DNA and/or RNA, oroligonucleotides, via either enzymatic or chemical synthesis to producefluorescent oligonucleotides. U.S. Pat. No. 5,135,717 describesphthalocyanine and tetrabenztriazaporphyrin reagents for use asfluorescent labels.

Detectable labels can be incorporated into nucleic acids by covalent ornon-covalent means, e.g., by transcription, such as by random-primerlabeling using Klenow polymerase, or nick translation, or,amplification, or equivalent as is known in the art. For example, in oneaspect, a nucleoside base is conjugated to a detectable moiety, such asa fluorescent dye, e.g., Cy3™ or Cy5™, and then incorporated intogenomic nucleic acids. Nucleic acids can be incorporated with Cy3™- orCy5™-dCTP conjugates mixed with unlabeled dCTP.

In another aspect, when using PCR or nick translation to label nucleicacids, modified nucleotides synthesized by coupling allylamine-dUTP tothe succinimidyl-ester derivatives of the fluorescent dyes or haptens(such as biotin or digoxigenin) can be used; this method allows custompreparation of most common fluorescent nucleotides, see, e.g.,Henegariu, Nat. Biotechnol 18:345-348, 2000.

Alternative non-covalent incorporation of label can be achieved usingother methods known in the art. For example, Kreatech Biotechnology'sUniversal Linkage System® (ULS®) provides a non-enzymatic labelingtechnology, wherein a platinum group forms a co-ordinative bond withDNA, RNA or nucleotides by binding to the N7 position of guanosine. Thistechnology may also be used to label proteins by binding to nitrogen andsulphur containing side chains of amino acids. See, e.g., U.S. Pat. Nos.5,580,990; 5,714,327; and 5,985,566; and European Patent No. 0539466.Thus, this system provides a method of associating any detectable labelwith members of a nucleic acid population, either directly into anucleic acid or peptide molecule associated thereto, or indirectly via acomplementary nucleic acid molecule or other partner molecule.

Labeling with a detectable label also can include a nucleic acidattached to another biological molecule, such as a nucleic acid, e.g.,an oligonucleotide, or a nucleic acid in the form of a stem-loopstructure as a “molecular beacon” or an “aptamer beacon”. Molecularbeacons as detectable moieties are well known in the art; for example,Sokol (Proc. Natl. Acad. Sci. USA 95:11538-11543, 1998) synthesized“molecular beacon” reporter oligodeoxynucleotides with matchedfluorescent donor and acceptor chromophores on their 5′ and 3′ ends. Inthe absence of a complementary nucleic acid strand, the molecular beaconremains in a stem-loop conformation where fluorescence resonance energytransfer prevents signal emission. On hybridization with a complementarysequence, the stem-loop structure opens increasing the physical distancebetween the donor and acceptor moieties thereby reducing fluorescenceresonance energy transfer and allowing a detectable signal to be emittedwhen the beacon is excited by light of the appropriate wavelength. Seealso, e.g., Antony (Biochemistry 40:9387-9395, 2001), describing amolecular beacon comprised of a G-rich 18-mer triplex formingoligodeoxyribonucleotide. See also U.S. Pat. Nos. 6,277,581 and6,235,504.

Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi,Anal. Biochem. 294:126-131, 2001; Poddar, Mol. Cell. Probes 15:161-167,2001; Kaboev, Nucl. Acids Res. 28:E94, 2000. Aptamer beacons can adopttwo or more conformations, one of which allows ligand binding. Afluorescence-quenching pair is used to report changes in conformationinduced by ligand binding. See also, e.g., Yamamoto, Genes Cells5:389-396, 2000; Smimov, Biochemistry 39:1462-1468, 2000.

In a preferred embodiment, genomic nucleic acids are labeled using anoligonucleotide linkage. The genomic nucleic acids are first digestedinto fragments with a restriction enzyme (e.g., AluI); fragments arethen associated with a unique capture sequence using a bridgingoligonucleotide. When properly designed, the unique fragment ispositioned directly adjoining the end of a nucleic acid such thatligation can be used to obtain covalent linkage. Each fragment can thenbe labeled with a dendrimeric construct comprising an oligonucleotidewhich hybridizes to the unique capture sequence associated with eachfragment. The fragments of two or more samples of nucleic acids can belabeled via a unique capture sequence associated with each respectivesample. In an especially preferred embodiment, multiple copies of thedetectable label are attached to a dendrimer to achieve signalamplification. Preferably, the use of a dendrimer in the methods of thepresent invention allows more than 10, 20, 50, 100, or 200 fluorophoremolecules to be attached to the genomic acids. Labeling of the fragmentscan be prior to hybridization of two or more nucleic acid samples, orpreferably following hybridization to maximize signal intensity.

Alternatively, the genomic nucleic acid may be labeled via a peptide. Apeptide can be made detectable by incorporating predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies,transcriptional activator polypeptide, metal binding domains, epitopetags). A label may also be attached via a second peptide (such as on adendrimer construct as above) that interacts with the first peptide(e.g., S—S association).

In another embodiment, the genomic nucleic acid may be labeled via apeptide nucleic acid. The term “peptide nucleic acid” (or PNA) as usedherein refers to a molecule comprising bases or base analogs such aswould be found in natural nucleic acid, but attached to a peptidebackbone rather than the sugar-phosphate backbone typical of nucleicacids. The attachment of the bases to the peptide is such as to allowthe bases to base pair with complementary bases of nucleic acid in amanner similar to that of an oligonucleotide. These small molecules,also designated anti gene agents, stop transcript elongation by bindingto their complementary strand of nucleic acid (Nielsen et al.,Anticancer Drug Des. 8:53 63, 1993).

Indirect labeling may be performed prior to or preferably, afterhybridization to maximize signal intensity. In a preferred embodiment,the hybridized surface is contacted with a first complex containing adetectable label and a first entity, wherein the first complexselectively reacts with the nucleic acids of either the test sample orthe reference sample; and either simultaneously or subsequently with asecond complex containing the same detectable label and a second entity,wherein the second complex selectively reacts with the nucleic acids ofthe other sample. In one embodiment, the first complex or the secondcomplex may comprise a differential linkage of the detectable label,such that one sample may be subjected to selective removal of thedetectable label (i.e., a subtractive approach). Alternatively, inanother embodiment, the first complex and the second complex do notcomprise a differential linkage of the detectable label, but instead,are added one following the other (i.e., an additive approach).

In certain embodiments, isolated or purified molecules may be preferred.As used herein, the terms “isolated”, “purified” or “substantiallypurified” refer to molecules, either nucleic acid or amino acidsequences, that are removed from their natural environment, isolated orseparated, and are at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which they are naturallyassociated. An isolated molecule is therefore a substantially purifiedmolecule.

Hybridization

The methods of the present invention can incorporate all known methodsand means and variations thereof for carrying out comparative genomichybridization, see, e.g., U.S. Pat. Nos. 6,197,501; 6,159,685;5,976,790; 5,965,362; 5,856,097; 5,830,645; 5,721,098; 5,665,549;5,635,351; Diago, Am. J. Pathol. 158:1623-1631, 2001; Theillet, Bull.Cancer 88:261-268, 2001; Werner, Pharmacogenomics 2:25-36, 2001; Jain,Pharmacogenomics 1:289-307, 2000.

Generally, nucleic acid hybridizations comprise the following majorsteps: (1) immobilization of target nucleic acids; (2) pre-hybridizationtreatment to increase accessibility of target DNA, and to reducenonspecific binding; (3) hybridization of the mixture of nucleic acidsto the nucleic acid on the solid surface; (4) post-hybridization washesto remove nucleic acid fragments not bound in the hybridization and (5)detection of the hybridized nucleic acid fragments. If indirectdetection is used, an additional step of hybridization with the labeledagent (e.g. dendrimer) and washing is needed. The reagent used in eachof these steps and their conditions for use vary depending on theparticular application.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. A number of methods for removing and/ordisabling the hybridization capacity of repetitive sequences are known(see, e.g., WO 93/18186). For instance, bulk procedures can be used. Inmany genomes, including the human genome, a major portion of sharedrepetitive DNA is contained within a few families of highly repeatedsequences such as Alu. These methods exploit the fact that hybridizationrate of complementary sequences increases as their concentrationincreases. Thus, repetitive sequences, which are generally present athigh concentration will become double stranded more rapidly than othersfollowing denaturation and incubation under hybridization conditions.The double stranded nucleic acids are then removed and the remainderused in hybridizations. Methods of separating single from doublestranded sequences include using hydroxyapatite or immobilizedcomplementary nucleic acids attached to a solid support, and the like.Alternatively, the partially hybridized mixture can be used and thedouble stranded sequences will be unable to hybridize to the target.

Alternatively, unlabeled sequences which are complementary to thesequences whose hybridization capacity is to be inhibited can be addedto the hybridization mixture. This method can be used to inhibithybridization of repetitive sequences as well as other sequences. Forexample, Cot-1 DNA can be used to selectively inhibit hybridization ofrepetitive sequences in a sample. To prepare Cot-1 DNA, DNA isextracted, sheared, denatured and renatured. Because highly repetitivesequences reanneal more quickly, the resulting hybrids are highlyenriched for these sequences. The remaining single stranded (i.e.,single copy sequences) is digested with S1 nuclease and the doublestranded Cot-1 DNA is purified and used to block hybridization ofrepetitive sequences in a sample. Although Cot-1 DNA can be prepared asdescribed above, it is also commercially available (BRL).

Hybridization conditions for nucleic acids in the methods of the presentinvention are well known in the art. Hybridization conditions may behigh, moderate or low stringency conditions. Ideally, nucleic acids willhybridize only to complementary nucleic acids and will not hybridize toother non-complementary nucleic acids in the sample. The hybridizationconditions can be varied to alter the degree of stringency in thehybridization and reduce background signals as is known in the art. Forexample, if the hybridization conditions are high stringency conditions,a nucleic acid will bind only to nucleic acid target sequences with avery high degree of complementarity. Low stringency hybridizationconditions will allow for hybridization of sequences with some degree ofsequence divergence. The hybridization conditions will vary depending onthe biological sample, and the type and sequence of nucleic acids. Oneskilled in the art will know how to optimize the hybridizationconditions to practice the methods of the present invention.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. Withhigh stringency conditions, nucleic acid base pairing will occur onlybetween nucleic acids that have sufficiently long segment with a highfrequency of complementary base sequences.

Exemplary hybridization conditions are as follows. High stringencygenerally refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.High stringency conditions can be provided, for example, byhybridization in 50% formamide, 5×Denhardt's solution, 5×SSC (salinesodium citrate) 0.2% SDS (sodium dodecyl sulphate) at 42° C., followedby washing in 0.1×SSC, and 0.1% SDS at 65° C. Moderate stringency refersto conditions equivalent to hybridization in 50% formamide, 5×Denhardt'ssolution, 5×SSC, 0.2% SDS at 42° C., followed by washing in 0.2×SSC,0.2% SDS, at 65° C. Low stringency refers to conditions equivalent tohybridization in 10% formamide, 5×Denhardt's solution, 6×SSC, 0.2% SDS,followed by washing in 1×SSC, 0.2% SDS, at 50° C.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” For example, the sequence“5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-5”. Certainbases not commonly found in natural nucleic acids may be included in thenucleic acids of the present invention and include, for example, inosineand 7-deazaguanine. Complementarity need not be perfect; stable duplexesmay contain mismatched base pairs or unmatched bases. Those skilled inthe art of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

Complementarity may be “partial” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids. Either term may also be used in referenceto individual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid strand, in contrast orcomparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “homology” and “homologous” refers to a degree of identitybetween two sequences. There may be partial homology or completehomology. A partially homologous sequence is one that is less than 100%identical to another sequence. Preferably, homologous sequences have anoverall identity of at least 70% or at least 75%, more preferably atleast 80% or at least 85%, most preferably at least 90% or at least 95%.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature”. The melting temperature is the temperature at which asample of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41 (% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization, 1985).Other references (e.g., Allawi and SantaLucia, Biochemistry 36:10581-94,1997) include more sophisticated computations which take structural andenvironmental, as well as sequence characteristics into account for thecalculation of T_(m).

Uracil-DNA N-glycosylase

The enzyme uracil-DNA N-glycosylase (UNG) selectively degrades nucleicacid having uracil residues by removing the uracil base from the DNAbackbone leading to degradation of the abasic DNA (both single- anddouble-stranded) upon subsequent heating or change in pH.Experimentally, UNG has been used to control contamination in PCR-basedgenotyping and sequencing applications as it can distinguish DNAincorporated with dUTP instead of dTTP and degrade those DNA moleculescontaining UTP. Accordingly, UNG degrades only DNA having uracil residesand does not degrade DNA having no uracil residues. Inventors have foundthat UNG is effective in degrading and selectively removing nucleic acidhaving uracil residues that are hybridized to an array withoutsignificantly affecting DNA hybridized to the array that does not haveuracil residues (for example nucleic acid having thymine residues).

Arrays

Nucleic acids used in the methods of the present invention can beimmobilized to or applied to an array or “biochip”. The term “array” or“microarray” or “biochip” or “chip” as used herein refers to a pluralityof elements arranged onto a defined area of a substrate surface. Inpracticing the methods of the invention, any known array and/or methodof making and using arrays can be incorporated in whole or in part, orvariations thereof, as disclosed, for example, in U.S. Pat. Nos.6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston, Curr. Biol. 8:R171-R174, 1998; Schummer, Biotechniques23:1087-1092, 1997; Kern, Biotechniques 23:120-124, 1997; Solinas-Toldo,Genes, Chromosomes & Cancer 20:399-407, 1997; Bowtell, Nature GeneticsSupp. 21:25-32, 1999. See also published U.S. Patent Applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Arrays are generically a plurality of “target elements” or “spots,” eachtarget element containing a defined amount of one or more biologicalmolecules, e.g., polypeptides, nucleic acid molecules, or probes,immobilized at discrete locations on a substrate surface. In preferredembodiments, the plurality of spots comprises nucleic acid segments,immobilized at preferably at least about 50, at least about 100, atleast about 300, or at least about 500 discrete locations on thesurface. The plurality may comprise multiple repeats of the same nucleicacid segments to produce, e.g., duplicate spots, triplicate spots,quadruplicate spots, quintuplicate spots, etc.

The resolution of array-based CGH is primarily dependent upon thenumber, size and map positions of the nucleic acid elements within thearray, which are capable of spanning the entire genome. Each nucleicacid of interest to be immobilized may be contained within a nucleicacid vector (e.g., plasmids, cosmids. etc.), or an artificialchromosome, such as a bacterial artificial chromosome (BAC) or P-1derived artificial chromosome as is known in the art, which are capableof incorporating large inserts of nucleic acid. Typically, bacterialartificial chromosomes, or BACs, which can each accommodate on averageabout 150 kilobases (kb) of cloned genomic DNA, are used in theproduction of the array. Preferably, each nucleic acid segment ofinterest is between about 1,000 (1 kB) and about 1,000,000 (1 MB)nucleotides in length, more preferably between about 100,000 (100 kB)and 300,000 (kB) nucleotides in length. Nucleic acid segments ofinterest may be chosen to span (i.e. collectively represent) thesequence of at least one chromosome, spaced at intervals along thechromosome (i.e. containing segments of chromosomal sequence) of about3-4 megabases (MB), more preferably at intervals of about 2-3 megabasesalong the chromosome, most preferably at intervals of about 1-2megabases along the chromosome. To represent the entire genomiccomplement, nucleic acid segments may be chosen to span all chromosomesat such intervals. Alternatively, selected genomic regions of interest,e.g., known mutational hotspots, may be selected from one or morechromosomes. Such genomic regions of interest may be nucleic acidsegments associated with a chromosomal abnormality, a contiguous geneabnormality, a genetically linked disease or syndrome.

Typically, the immobilized nucleic acid molecules are contacted with asample for specific binding, e.g., hybridization, between molecules inthe sample and the array. Immobilized nucleic acids segments can containsequences from specific messages (e.g., as cDNA libraries) or genes(e.g., genomic libraries), including, e.g., substantially all or asubsection of a chromosome or substantially all of a genome, including ahuman genome. Other target elements can contain reference sequences,such as positive and negative controls, and the like. The targetelements of the arrays may be arranged on the substrate surface atdifferent sizes and different densities. Different target elements ofthe arrays can have the same molecular species, but, at differentamounts, densities, sizes, labeled or unlabeled, and the like. Thetarget element sizes and densities will depend upon a number of factors,such as the nature of the label (the immobilized molecule can also belabeled), the substrate support (it is solid, semi-solid, fibrous,capillary or porous), and the like.

Each target element may comprise substantially the same nucleic acidsequences, or, a mixture of nucleic acids of different lengths and/orsequences. Thus, for example, a target element may contain more than onecopy of a cloned piece of DNA, and each copy may be broken intofragments of different lengths, as described herein. The length andcomplexity of the nucleic acid fixed onto the array surface is notcritical to the invention. The array can comprise nucleic acidsimmobilized on any substrate, e.g., a solid surface (e.g.,nitrocellulose, glass, quartz, fused silica, plastics and the like).See, e.g., U.S. Pat. No. 6,063,338 describing multi-well platformscontaining cycloolefin polymers if fluorescence is to be measured.Arrays used in the methods of the invention can comprise housingcontaining components for controlling humidity and temperature duringthe hybridization and wash reactions.

The CGH methods of the invention can be performed using any type ofarray. Commercially available CGH arrays or prepared slides for arrayprinting include, for example, GeneChips™ from Affymetrix, Santa Clara,Calif.; Spectral Chip™ Mouse BAC Arrays and Spectral Chip™ Human BACArrays and other custom Arrays from Spectral Genomics, Houston, Tex.;Codelink™ Human Bioarrays from Amersham Biosciences (GE Healthcare); andUltraGap™ from Dow Corning, Elizabethtown, Ky. UltraGap™ slides used inaccordance with the manufacturer's suggested protocol are preferred.

In a preferred embodiment, the surface comprises an array containingone, several or all of the human genomic nucleic acid segments providedin a compendium of bacterial artificial chromosomes (BACs) compiled byThe BAC Resource Consortium, and referred to in the art by their RPI orCTB clone names, see Cheung et al., Nature 409:953-958, 2001. Thiscompendium contains 7,600 cytogenetically defined landmarks on the draftsequence of the human genome (see McPherson et al., Nature 409:934-41,2001). These landmarks are large-insert clones mapped to chromosomebands by fluorescence in situ hybridization, each containing a sequencetag that is positioned on the genomic sequence. These clones representall 24 human chromosomes in about 1 Mb resolution. Sources of BACgenomic collections include the BACPAC Resources Center(CHORI—Children's Hospital Oakland Research Institute), ResGen (ResearchGenetics through Invitrogen) and The Sanger Center (UK).

Many methods for immobilizing nucleic acids on a variety of solidsurfaces are known in the art. For instance, the solid surface may be amembrane, glass, plastic, or a bead. The desired component may becovalently bound or noncovalently attached through nonspecific binding.The immobilization of nucleic acids on solid surfaces is discussed morefully below.

A wide variety of organic and inorganic polymers, as well as othermaterials, both natural and synthetic, may be employed as the materialfor the solid surface Illustrative solid surfaces includenitrocellulose, nylon, glass, diazotized membranes (paper or nylon),silicones, polyformaldehyde, cellulose, and cellulose acetate. Inaddition, plastics such as polyethylene, polypropylene, polystyrene, andthe like can be used. Other materials which may be employed includepaper, ceramics, metals, metalloids, semiconductive materials, cermetsor the like. In addition substances that form gels can be used. Suchmaterials include proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides. Where the solid surface isporous, various pore sizes may be employed depending upon the nature ofthe system.

In preparing the surface of a solid support for array printing, aplurality of different materials may be employed, particularly aslaminates, to obtain various properties. For example, proteins (e.g.,bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt'ssolution) can be employed to avoid non-specific binding, simplifycovalent conjugation, enhance signal detection or the like.

If covalent bonding between a compound and the surface is desired, thesurface may be polyfunctional or be capable of being polyfunctionalized.Functional groups which may be present on the surface and used forlinking can include carboxylic acids, aldehydes, amino groups, cyanogroups, ethylenic groups, hydroxyl groups, mercapto groups and the like.The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature. For example,methods for immobilizing nucleic acids by introduction of variousfunctional groups to the molecules is known (see, e.g., Bischoff et al.,Anal. Biochem. 164:336-344, 1987); Kemsky et al., Nucl Acids Res.15:2891-2910, 1987). Modified nucleotides can be placed on the targetusing PCR primers containing the modified nucleotide, or by enzymaticend labeling with modified nucleotides.

Alternative surfaces include derivatized surfaces such as chemicallycoated glass slides. On example, is the CodeLink™ Activated Slide fromAmersham Biosciences. These slides are coated with a novel 3-D surfacechemistry comprised of a long-chain, hydrophilic polymer containingamine-reactive groups, to react with and covalently immobilizeamine-modified DNA for microarrays. This polymer is covalentlycrosslinked to itself and to the surface of the slide and is designed toorient the immobilized DNA away from the surface of the slide to improvehybridization. Another such 3D slide is UltraGap™, sold by Dow Corning.

Use of membrane supports (e.g., nitrocellulose, nylon, polypropylene)for the nucleic acid arrays of the invention is advantageous because ofwell developed technology employing manual and robotic methods ofarraying targets at relatively high element densities (e.g., up to30-40/cm²). In addition, such membranes are generally available andprotocols and equipment for hybridization to membranes is well known.Many membrane materials, however, have considerable fluorescenceemission, where fluorescent labels are used to detect hybridization.

To optimize a given assay format one of skill can determine sensitivityof fluorescence detection for different combinations of membrane type,fluorophore, excitation and emission bands, spot size and the like. Inaddition, low fluorescence background membranes have been described(see, e.g., Chu et al., Electrophoresis 13:105-114, 1992).

The sensitivity for detection of spots of various diameters on thecandidate membranes can be readily determined by, for example, spottinga dilution series of fluorescently end labeled DNA fragments. Thesespots are then imaged using conventional fluorescence microscopy. Thesensitivity, linearity, and dynamic range achievable from the variouscombinations of fluorophore and membranes can thus be determined. Serialdilutions of pairs of fluorophore in known relative proportions can alsobe analyzed to determine the accuracy with which fluorescence ratiomeasurements reflect actual fluorophore ratios over the dynamic rangepermitted by the detectors and membrane fluorescence.

Arrays on substrates with much lower fluorescence than membranes, suchas glass, quartz, or small beads, can achieve much better sensitivity.For example, elements of various sizes, ranging from about 1 mm diameterdown to about 1 μm can be used with these materials. Small array memberscontaining small amounts of concentrated target DNA are convenientlyused for high complexity comparative hybridizations since the totalamount of probe available for binding to each element will be limited.Thus, it is advantageous to have small array members that contain asmall amount of concentrated target DNA so that the signal that isobtained is highly localized and bright. Such small array members aretypically used in arrays with densities greater than 10⁴/cm². Relativelysimple approaches capable of quantitative fluorescent imaging of 1 cm²areas have been described that permit acquisition of data from a largenumber of members in a single image (see, e.g., Wittrup et al.,Cytometry 16:206-213, 1994).

Covalent attachment of the target nucleic acids to glass or syntheticfused silica can be accomplished according to a number of knowntechniques. Such substrates provide a very low fluorescence substrate,and a highly efficient hybridization environment.

There are many possible approaches to coupling nucleic acids to glassthat employ commercially available reagents. For instance, materials forpreparation of silanized glass with a number of functional groups arecommercially available or can be prepared using standard techniques.Alternatively, quartz cover slips, which have at least 10-fold lowerauto fluorescence than glass, can be silanized.

The targets can also be immobilized on commercially available coatedbeads or other surfaces. For instance, biotin end-labeled nucleic acidscan be bound to commercially available avidin-coated beads. Streptavidinor anti-digoxigenin antibody can also be attached to silanized glassslides by protein-mediated coupling, using e.g., protein A followingstandard protocols (see, e.g., Smith et al., Science 258:1122-1126,1992). Biotin or digoxigenin end-labeled nucleic acids can be preparedaccording to standard techniques.

Hybridization to nucleic acids attached to beads is accomplished bysuspending them in the hybridization mix, and then depositing them onthe glass substrate for analysis after washing. Alternatively,paramagnetic particles, such as ferric oxide particles, with or withoutavidin coating, can be used.

Interpretation of Array-Based CGH

The copy number of particular nucleic acid sequences in a test sampleand a reference sample are compared by hybridizing the samples to one ormore target nucleic acid segments. The hybridization signal intensity,and the ratio of intensities, produced by the detectable labelassociated with each sample is determined. Typically, the greater theratio of the signal intensities on a target nucleic acid segment, thegreater the copy number ratio of sequences in the two samples that bindto that element. Thus comparison of the signal intensity ratios amongtarget nucleic acid segments permits comparison of copy number ratios ofdifferent sequences in the genomic nucleic acids of the two samples.

In addition to labeling nucleic acids with fluorescent dyes, theinvention can be practiced using any apparatus or methods to detectdetectable labels associated with nucleic acids of a sample, anindividual member of the nucleic acids of a sample, or anarray-immobilized nucleic acid segment, or, any apparatus or methods todetect nucleic acids specifically hybridized to each other. Devices andmethods for the detection of multiple fluorophores are well known in theart, see, e.g., U.S. Pat. Nos. 5,539,517; 6,049,380; 6,054,279;6,055,325; and 6,294,331. Any known device or method, or variationthereof, can be used or adapted to practice the methods of theinvention, including array reading or “scanning” devices, such asscanning and analyzing multicolor fluorescence images; see, e.g., U.S.Pat. Nos. 6,294,331; 6,261,776; 6,252,664; 6,191,425; 6,143,495;6,140,044; 6,066,459; 5,943,129; 5,922,617; 5,880,473; 5,846,708;5,790,727; and, the patents cited in the discussion of arrays, herein.See also published U.S. Patent Application Nos. 20010018514;20010007747; and published international patent applications Nos.WO0146467 A; WO9960163 A; WO0009650 A; WO0026412 A; WO0042222 A;WO0047600 A; and WO0101144 A.

For example a spectrograph can image an emission spectrum onto atwo-dimensional array of light detectors; a full spectrally resolvedimage of the array is thus obtained. Photophysics of the fluorophore,e.g., fluorescence quantum yield and photodestruction yield, and thesensitivity of the detector are read time parameters for anoligonucleotide array. With sufficient laser power and use of Cy5™ orCy3™, which have lower photodestruction yields an array can be read inless than 5 seconds.

Charge-coupled devices, or CCDs, are used in microarray scanningsystems, including practicing the methods of the invention. Colordiscrimination can also be based on 3-color CCD video images; these canbe performed by measuring hue values. Hue values are introduced tospecify colors numerically. Calculation is based on intensities of red,green and blue light (RGB) as recorded by the separate channels of thecamera. The formulation used for transforming the RGB values into hue,however, simplifies the data and does not make reference to the truephysical properties of light. Alternatively, spectral imaging can beused; it analyzes light as the intensity per wavelength, which is theonly quantity by which to describe the color of light correctly. Inaddition, spectral imaging can provide spatial data, because it containsspectral information for every pixel in the image. Alternatively aspectral image can be made using brightfield microscopy, see, e.g., U.S.Pat. No. 6,294,331.

A specific advantage of the methods of the present invention is that asingle detectable label may be used. This eliminates the need to readand co-ordinate multiple colored fluorophores. Thus, signal intensity atthe lower range is uniform and can readily be normalized, as opposed tohaving to account for differences in signal intensity amongst more thanone fluorophore. Other advantages of the present invention's array-basedCGH approach include the increased resolution by spanning across theentire genomic sequence of each chromosome and the increased sensitivityachieved as compared to traditional in situ chromosomal hybridization.

The methods of the invention further comprise data analysis, which caninclude the steps of determining, e.g., fluorescent intensity as afunction of substrate position, removing “outliers” (data deviating froma predetermined statistical distribution), or calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with color in each region varyingaccording to the light emission or binding affinity between targets andprobes. See, e.g., U.S. Pat. Nos. 5,324,633; 5,863,504; and 6,045,996.The invention can also incorporate a device for detecting a labeledmarker on a sample located on a support, see, e.g., U.S. Pat. No.5,578,832.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Preparation of a Genomic Nucleic Acid Array

A variety of microarray equipment (e.g., BioRobotics Microgrid andothers; collectively “arrayers”) are available for printing the nucleicacid material onto a plurality of discrete locations of a solid surface.Two specific surfaces were printed with native BAC DNA to establish aprotocol for the specific application of large-insert clone microarrayfabrication (e.g., BACs, PACs, cosmids).

Typical prior art arrayer installation and validation protocols assessthe printing performance of an arrayer using either dye-only solutionsor dye-oligo DNA solutions. These conditions do not reflect the fluiddynamics associated with large clone array manufacturing and hence aresub-optimal for generating printing parameters. The present exampledescribed herein establishes a simple and qualitative approach tovalidating arrayers and establishing printing parameters for largeinsert clone microarray fabrication.

A sample collection of the large insert DNA clones (BACs, PACs, cosmids)intended for printing was resuspended in a salt containing printingbuffer (e.g., 50-150 mM sodium phosphate, pH 8-9) at a concentration of75-100 ng/μl. The DNA was briefly fragmented using an ultrasonicwater-bath processor set at 100 A with 70 W output for 5 seconds. Gelelectrophoreses (0.8-1.0% agarose) was used to confirm that the size ofthe fragmented DNA ranged homogenously within 500 base pairs and larger.To a 30 μl aliquot of the sonicated DNA was added 1 μl of fluorescentnucleotide dye-conjugate (1 mM) of choice. Samples were mixed andtransferred to a printing surface. Upon completion of the printingprocess, the resulting image was evaluated by scanning with a laserscanner (e.g., Axon 4000, 4100, 4200) set at the wavelength offluorescent dye used.

Under these typical parameters, two surfaces were tested. The firstsurface was plain glass slides cleaned according to a standard base/acidprotocol. Fluorescent measurements on plain glass slides indicated abackground reading of about 3000, with a spot intensity of about 10,000,and a spot size of approximately 290 μm. The second surface was theCodeLink™ Activated Slide (Amersham Biosciences). Fluorescentmeasurements on the CodeLink™ Activated Slide indicated a backgroundreading of about 15,000, with a spot intensity of about 65,000, and aspot size of approximately 180 μm.

EXAMPLE 2 Preparation of Genomic Nucleic Acids

Labeling. Genomic DNA may be labeled by any standard protocol toincorporate a detectable label. An exemplary random priming with afluorophore is as follows. In a 100 μl reaction containing 1 ng to 1 μgDNA, combine 1× random primers solution (BioPrime DNA Labeling System,Gibco BRL), 1 mM Tris, pH 7.6, 0.1 mM EDTA, 0.2 mM each of dATP, dTTPand dGTP, 0.1 mM dCTP, 0.4 mM Cy3 or Cy5-dCTP (Amersham) and 160 UKlenow fragment (BioPrime DNA Labeling System, Gibco BRL). The DNA andrandom primers solution is incubated at 100° C. for 10 minutes in atotal volume of 84 μl, prior to adding the other reagents, and then thefinal 100 μl reaction is incubated overnight at 37° C. Unincorporatednucleotides are removed using a Sephadex G-50 column.

Dendrimeric labeling. Genomic DNA may contain a tag contained within adendrimeric construct. A dendrimer is a highly branched molecule createdto integrate multiple copies of the desired detectable label to amplifydetection. Kits for dendrimer labeling and construction are commerciallyavailable (e.g., Genisphere Inc.). Briefly, genomic DNA is digested withAluI to yield digested fragments of about 256 bp on average. The genomicDNA fragments are then treated with 3′ TdT to attach a poly-T tail toeach fragment. A ligation containing (i) a bridging oligonucleotide witha poly-A tail, (ii) a capture sequence oligonucleotide (with one endcomplementary to the bridging oligonucleotide), and (iii) the T-tailedfragments is then performed, resulting in each genomic DNA fragmenthaving the same unique capture sequence at its 3′ end. Each sample ofgenomic DNA (i.e., the test and the reference samples of nucleic acids)is coupled to a unique capture sequence prior to hybridization.Following hybridization, the genomic DNA fragments can then be labeledusing a dendrimer that contains an oligonucleotide complementary to theunique capture sequence of a one sample and multiple copies of label,typically fluorescent dye molecules.

Alternatively, genomic mRNA is first reverse transcribed with unlabelleddATP, dTTP, dGTP and dCTP using a primer oligonucleotide that contains aunique capture sequence and a poly-T tail to hybridize to the poly-Atail of the mRNA molecules. The reaction is then stopped and the mRNA isdegraded to yield genomic cDNAs containing the unique capture sequence.These genomic cDNAs can then be labeled using a dendrimer that containsan oligonucleotide complementary to the unique capture sequence andmultiple copies of label, typically fluorescent dye molecules.Genisphere, Inc. offers a variety of dendrimers that vary in size andfluorescence intensity. The Array 900 and 350 series kits containfour-layer dendrimers. A four layer dendrimer theoretically has 324single stranded DNA arms in the outer layer. The diameter of a fourlayer dendrimer is 182-190 nm and the molecular weight is 1.2×10⁷Daltons. The Array 50 series kit contains a two layer dendrimer. A twolayer dendrimer theoretically has 45 single stranded DNA arms in theouter layer. The diameter of a two layer dendrimer is 70-90 nm and themolecular weight is 1.3×10⁶ Daltons.

EXAMPLE 3 Comparative Genomic Hybridization

Genomic nucleic acids obtained from a test sample and a referencesample, each population containing a unique capture sequence, arecombined (about 1-2 μg each) with Cot-1 DNA (80-100 μg) and precipitatedwith ethanol. Precipitate is collected by centrifugation and allowed toair dry for 10 minutes before re-dissolving it in a 50 μl hybridizationmixture containing 50% formamide, 2×SSC, 10% dextran sulfate, 4% SDS and500 μg yeast tRNA, pH 7. The hybridization mixture is incubated at 70°C. for 10-15 minutes to denature the DNA and subsequently at 37° C. for60 minutes to allow blocking of repetitive sequences. To the array isadded 50 μl of slide blocking solution containing 500 μg salmon spermDNA in 50% formamide, 2×SSC, 10% dextran sulfate and 4% SDS, pH 7. Aftera 30 minute incubation at room temperature, approximately three-quartersof the blocking solution is removed, and the denatured and re-annealedhybridization mixture is added and hybridized at 37° C. for 16-72 hours.After hybridization, excess hybridization fluid is rinsed off with 0.1 Msodium phosphate, 0.1% NP40, pH 8, then the array is washed once in 50%formamide, 2×SSC, pH 7 at 45° C. for 15 minutes, and finally with 0.1 Msodium phosphate, 0.1% NP40, pH 8 at room temperature for 15 minutes.

EXAMPLE 4 Single Label CGH with Subtractive Labeling

An exemplary selective removal can be achieved by making the labelassociated with either the genomic nucleic acids obtained from the testsample or the genomic nucleic acids obtained from the reference samplesusceptible to removal with atmospheric ozone. Certain fluorophores(e.g., Cy5™ and Alexa 647) are susceptible to ozone levels as low asabout 5-10 ppm for periods as short as 10-30 seconds. Followinghybridization, arrays are placed in an enclosed chamber with an ozonegenerator to achieve at atmospheric ozone level of about 60-85 ppm forabout 10-30 minutes. Selective removal of the label from one populationof genomic nucleic acids may be achieved by modifying the physicalnature of the labeling process, such as increasing the distance of thelabel from the genomic DNA to increase exposure to the atmosphericozone.

Another exemplary selective removal can be achieved by making the labelassociated with either the genomic nucleic acids obtained from the testsample or the genomic nucleic acids obtained from the reference samplesusceptible to removal by cleavage with a restriction endonuclease or ahoming endonuclease. In this example, reference sample genomic nucleicacids are prepared with a first unique capture sequence to which islinked a dendrimer containing an oligonucleotide complementary to thisfirst unique capture sequence and a fluorescent label. The test samplegenomic nucleic acids are prepared with a second unique capture sequencecontaining a stretch of nucleotides representing the recognitionsequence for an endonuclease to which is linked a dendrimer containingan oligonucleotide complementary to this second unique capture sequenceand the same fluorescent label as used for the first sample. Followinghybridization of the test and reference genomic nucleic acids to anarray containing a plurality of immobilized nucleic acid segments ofinterest, the fluorescence of the array is measured.

The array is then contacted with the endonuclease recognizing thesequence contained within the second unique capture sequence underconditions allowing cleavage of the dendrimeric construct from thegenomic nucleic acids to selectively remove the fluorescent label fromthe test sample nucleic acids.

Another exemplary selective removal can be achieved by making the labelassociated with either the genomic nucleic acids obtained from the testsample or the genomic nucleic acids obtained from the reference samplesusceptible to removal by UV irradiation. The label is incorporatedusing a linker that is photocleavable, such as a linker containing a2-nitrobenzyl group (see, e.g., Bai et al., Proc. Natl. Acad. Sci.100:409-413, 2003). Following hybridization, arrays are placed in achamber with water and irradiated with a UV lamp at 340 nm (lightintensity of about 20 mW/cm²) for about 5-10 minutes to selectivelyremove the label from one population of genomic nucleic acids only(i.e., the nucleic acids containing the photocleavable linker).

Thus, in these examples of selective removal, data from the array isacquired at two time points, with the same fluorophore being read. Thefirst acquisition is after the comparative genomic hybridization (e.g.,before the selective removal of the label from the test sample genomicnucleic acids), in part to determine the fluorescence of the combinednucleic acid samples (F_(Total)). The second acquisition is after theselective removal of the label, in part to determine the remainingfluorescence of the reference sample genomic nucleic acids(F_(Reference)). The fluorescence of the test sample genomic nucleicacids (F_(Test)) is then equal to (F_(Total)−F_(Reference)). Thus, thesame fluorophore can be used to achieve maximal uniformity between thetwo genomic nucleic acid samples, and between tests performed withdifferent samples. If the selective removal is designed to removenucleic acid associated with the reference genomic DNA, then the secondread would be F_(test) and the difference between F_(Test) and F_(Total)would be F_(Reference).

As a quality control in single label CGH the two linkers for the testand reference labels are switched and comparative hybridizationrepeated.

EXAMPLE 5 Single Label CGH with Additive Labeling

Exemplary additive labeling for single label CGH can be achieved byperforming a first comparative hybridization wherein the genomic nucleicacids obtained from the reference sample comprise a first uniqueoligonucleotide tag and the genomic nucleic acids obtained from the testsample comprise a second unique oligonucleotide tag. Followinghybridization of the test and reference genomic nucleic acids to anarray containing a plurality of immobilized nucleic acid segments ofinterest, the array is exposed to a first dendrimeric complex containingan oligonucleotide complementary to the first unique oligonucleotide tagand a fluorescent label. This provides a selective labeling of thereference sample genomic nucleic acids.

Preferred conditions for dendrimer hybridization include use of Pronto!™hybridization buffer (Corning, Inc.) with 50 μg of Cot 1 DNA and 50-100μg of SST (shredded (sonicated) salmon testis DNA). Cot 1 DNA may bereplaced by any other non-mammalian genomic DNA such as plant DNA, fishDNA, bacterial DNA, and non-natural DNA, e.g. dendrimeric DNA. After 30min. hybridization, the array is washed as follows:

-   -   1. Soak slide in 2×SSC containing 0.01% SDS (pH 7.5-8.0) at room        temperature until coverslip is loosened (<3 minutes).    -   2. Incubate for 5 min. with gentle agitation at 50 C in 2×SSC        containing 0.01% SDS (pH 7.5-8.0).    -   3. Incubate for 5 min. with gentle agitation at room temperature        in 2×SSC (pH 7.5-8.0).    -   4. Incubate for 5 min. with gentle agitation at room temperature        in 0.2×SSC (pH 7.5-8.0).        -   SDS: sodium doedecyl sulfate (detergent)        -   1×SSC: 0.15 molar sodium chloride and 0.015 molar sodium            citrate

Data from the array is then acquired, in part to determine thefluorescence of the first reference sample genomic nucleic acids(F_(Reference)). The array is then exposed to a second dendrimericcomplex containing an oligonucleotide complementary to the second uniqueoligonucleotide tag and the same fluorescent label as used in the firstdendrimeric complex. Data from the array is then acquired for a secondtime, in part to determine the fluorescence of the combined nucleicacids (F_(Total)). The fluorescence of the test sample genomic nucleicacids (F_(Test)) is then equal to (F_(Total)−F_(Reference)). Thus, thesame fluorophore can be used to achieve maximal uniformity between thetwo genomic nucleic acid samples, and between tests performed withdifferent samples. If the first dendrimeric complex binds to F_(test),then the difference between F_(Test) and F_(Total) would beF_(Reference).

As a quality control in single label CGH the unique tag sequencesattached to the test and reference genomic nucleic acids are switchedand comparative hybridization repeated.

EXAMPLE 6 Single Label CGH with Subtractive Labeling Using UNG

For the purposes of this example male genomic DNA was used as the “testsample” and female genomic DNA was used as the “reference sample.”

Solution Preparation

-   -   dTTP Labeling Buffer: The following dNTPs were added to 47.3 mL        of sterile water, 500 mL Tris-HCl pH 7.5, and 100 μL of 0.5M        EDTA: 600 μL of 100 mM dATP, 300 μL of 100 mM dCTP, 600 μL of        100 mM dGTP and 600 μL of 100 mM dTTP.    -   dTTP Labeling Mix: 5 μL of dTTP Labeling Buffer was mixed with        1.0 μL Cy3-dCTP (Amersham Cat. # PA53031) and 1 μL exo-Klenow        (Invitrogen Cat. # 18095-012).    -   dUTP Labeling Buffer: The following dNTPs were added to 47.3 mL        of sterile water, 500 mL Tris-HCl pH 7.5, and 100 μL of 0.5M        EDTA: 600 μL of 100 mM dATP, 300 μL of 100 mM dCTP, 600 μL of        100 mM dGTP and 600 μL of 100 mM dUTP.    -   dUTP Labeling Mix: 5 μL of dUTP Labeling Buffer was mixed with        1.0 μL Cy3-dCTP (Amersham Cat. # PA53031) and 1 μL exo-Klenow        (Invitrogen Cat. # 18095-012).    -   Binding Buffer B2: (Invitrogen Cat. # K3100-2)    -   Wash Buffer W1: (Invitrogen Cat. # K3100-2)    -   UNG Digestion Solution: A volume of 3 μL of Uracil DNA        glycosylase (UNG; Invitrogen Catalogue number 18054-015) and a        volume of 3 μL of 10×PCR Buffer (Qiagen Cat. # 201203) were        added to 24 μL sterile water.

Amplification Reaction

Test sample DNA and reference sample DNA (2 μg each) were diluted tofinal volumes of 22 μL and mixed gently. From the test sample DNA, 11 μLwas aliquoted into a tube labeled “Test_(dUTP)” and 11 μL was aliquotedinto a tube labeled “Test_(dTTP)”; and from the reference sample DNA, 11μL was aliquoted into a tube labeled “Referenced_(dUTP)” and 11 μL wasaliquoted into a tube labeled “Referenced_(dTTP)”. Next, 12 μL of RandomPrimers (Invitrogen Cat. # 18095-012) were added to each of the“Test_(dUTP)”; “Test_(dTTP)”; “Reference_(dUTP)” and “Referenced_(dTTP)”tubes; the tubes were vortexed, centrifuged; denatured by heating 5 minat 99° C.; and then snap cooled on an ice slurry for 5 min.

Next, dTTP Labeling Mix (7 μL) was added to the “Test_(dTTP)” and“Referenced_(dTTP)” tubes; dUTP Labeling Mix (7 μL) was added to the“Test_(dUTP)” and “Reference_(dUTP)”; and the nucleic acids in the eachof the tubes were amplified by incubating the tubes in the dark for 2hours at 37° C. Next 3 μL of 0.5M EDTA was added to each of theamplifications reactions and the tubes were vortexed and centrifuged.

CGH Reactions

The amplified and labeled products of the “Test_(dUTP)” and“Referenced_(dTTP)” were combined and the amplified and labeled productsof the “Test_(dTTP)” and “Reference_(dUTP)” were also combined.

The combined products were then purified using the PureLink PCRPurification System as follows: Binding Buffer B2 (400 μL) was added toeach tube of the combined products; and the samples were loaded on toPureLink Spin 1 column; the columns were centrifuged ate 10,000 g for 1min and the flow through was discarded; 650 μL of Wash Buffer W1 wasadded to each column; centrifuged ate 10,000 g for 1 min and the flowthrough was discarded; and the columns were re-centrifuged at high speedfor an additional 2-3 minutes to remove any residual wash buffer. Thecolumns were then transferred to Eppendorf tubes; 50 μL of sterile waterwas added to the columns and the columns were allowed to sit for 1minute; the columns were centrifuged for 2 minutes at high speed and theflow through containing the purified combined nucleic acid productssamples were saved.

The purified combined nucleic acid products were then precipitated togenerate hybridization solutions as follows: 50 μg of human Cot-1 DNAand 100 μg of Salmon Sperm DNA were added to each of the solutions andthe solutions were vortexed; a volume of 5M NaCl that was 1/12 of thetotal solution volume was added to each of the solutions and thesolutions were vortexed; a volume of isopropanol that was 75% the totalsolution volume was added to each of the solutions and the solutionswere vortexed; the solutions were left to sit at room temperature for 20minutes and then centrifuged at high speed for 20 minutes; thesupernatant was aspirated and the pellet was washed with 700 μL of 70%ethanol; the pellet was allowed to air-dry for 10 minutes; 20 μL ofPronto! Long Oligo Hybridization Solution (from Pronto! UniversalHybridization Kit) was added to each tube; the tubes were heated on aheat block at 95° C. for 2.5 minutes the tubes were vigorously vortexedfor 15-30 seconds to dissolve the pellet and the tubes were placed backon the heating block at 95° for an additional 2.5 minutes; the tubeswere vortexed vigorously and centrifuged at high speed for 2 minutes.

The precipitated hybridization samples were then hybridized to human BACCGH arrays having 1296 clones printed in triplicate on aminosilanecoated slides (Corning). Hybridization was performed as follows: 19 μLof the hybridization solutions were pippetted onto the individual arrayswith the Test_(dUTP)/Referenced_(dTTP) combined solution applied to thetop array and the Test_(dTTP)/Reference_(dTTP) applied to the bottomarray; a cover slip was placed on each array to allow dispersion of thesolution; 10 μl of de-ionized water was loaded onto each well of ahybridization chamber (Corning; catalog number 2551); the arrays wereplaced into the hybridization chamber and the chamber was sealed; thehybridization chambers were wrapped in foil; all components were sealedin a plastic bag with a moist paper towel; and the entire package wasplaced in a 42° C. incubator overnight. The next day the hybridizedarrays were washed using standard genomic wash procedures and the arrayswere baked at 80° C. for 30 minutes.

The hybridized arrays were then scanned using a an Axon 4200B scanner(Scan-1).

Following the scan the arrays were treated with UNG to selectivelydegrade and remove nucleic acids having uracil residues as follows: 30μL of UNG Digestion Solution was pipetted onto the arrays; a LifterSlip(Fisher catalogue number 24X30I-2-5111-001-LS) was placed on the arrayto displace the solution; 10 μl of de-ionized water was loaded onto eachwell of a hybridization chamber (Corning; catalog number 2551); thearrays were placed into the hybridization chamber and the chamber wassealed; the hybridization chambers were wrapped in foil; thehybridization chambers were placed in a incubator at 37° C. for 1 hour;the hybridization chambers transferred to a 70° C. oven for 15 min; andthe hybridized arrays were washed using standard genomic washprocedures.

Following the UNG treatment, the arrays were re-scanned using a an Axon4200B scanner (Scan-2).

The array data for the CGH arrays were analyzed and calculated usingArray CGH software developed by InfoQuant. Differences between the Testand Reference nucleic acids were calculated using the following formulafor CGH where the reference nucleic acids were amplified with dTTP andtest nucleic acids were amplified with dUTP:(Scan-1−Scan-2)/Scan-2=Test/Reference. Differences between the Test andReference nucleic acids were calculated using the following formula forCGH where the reference nucleic acids were amplified with dUTP and testnucleic acids were amplified with dTTP:(Scan-1−Scan-2)/Scan-2=Reference/Test.

Effect of UNG on Nucleic Acid Hybridized to the Array. Reference nucleicacid was amplified and hybridized to arrays in accordance with theprocedures described above in this example, with the exception that notest samples were used in the reaction. Specifically, reference DNA wasamplified using dTTP and was amplified using dUTP. The amplified dUTPnucleic acid was applied to the top array and the dTTP nucleic acid wasapplied to the bottom array. The arrays were scanned, treated with UNGand then re-scanned. In the first scan, there was no significantdifference between the reference DNA amplified using dTTP and thatamplified using dUTP. In the second scan, the signal from the referenceDNA amplified using dUTP was reduced to nearly undetectable levelsindicating that the UNG had effectively degraded and removed from thearray the uracil containing nucleic acids. In contrast to the DNAamplified using dUTP, in the array hybridized with reference nucleicamplified using dTTP there was no detectable difference in the signalfrom the first scan and the signal from the second scan performed afterthe UNG degradation. Accordingly, this example demonstrates that UNG iseffective in selectively removing uracil containing uracil-containingnucleic acid from the array without having any significant effect onnon-uracil-containing nucleic acid hybridized to the array.

Results of Single-Label UNG-Subtractive CGH Using Male and FemaleGenomic DNA. The results of CGH performed using normal male genomic DNAas a test sample and normal female genomic DNA a the reference sample asdescribed in this example clearly demonstrated a loss of test (male)nucleic acid associated with the X-chromosome and an increase in nucleicacid associated with the Y-chromosome. There were no notable differencesin the amount of nucleic acids associated with chromosomes other thanthe sex chromosomes.

EXAMPLE 7 Detection of a Chromosomal Deletion Using Single-LabelUNG-Subtractive CGH

CGH was performed as described in Example 6 using genomic DNA from afemale known to have a deletion in chromosome 3 (46,XX,del(3)(p12p21.1))as the test sample and normal male genomic DNA as the reference sample.The results clearly demonstrated an increase of test nucleic acidassociated with the X-chromosome and an decrease in nucleic acidassociated with the Y-chromosome and in nucleic acid in the locus of thechromosome 3 where the known deletion was. There were no other notabledifferences in the amount of nucleic acids between the samples.

EXAMPLE 8 Detection of a Chromosomal Translocation Using Single-LabelUNG-Subtractive CGH

CGH was performed as described in Example 6 using genomic DNA from amale known to have a translocation (ish XY,SubTel der(3)t(3p−;16q+)) asthe test sample and normal female genomic DNA as the reference sample.The results clearly allowed for the detection of the translocation,i.e., the loss in both chromosome 3 and the gain in chromosome 16 in thetest sample as compared to the reference sample, as well as thedifferences in the X and Y chromosomes. There were no other notabledifferences in the amount of nucleic acids between the samples.

EXAMPLE 9 Detection of a Chromosomal Deletion Using Single-LabelUNG-Subtractive CGH

CGH was performed as described in Example 6 using genomic DNA from amale known to have a deletion in chromosome 11 (46,XY,del(11)(q23q23))as the test sample and normal female genomic DNA as the referencesample. The results clearly allowed for the detection of the deletion,i.e., the loss of chromosome 11 in the test sample as compared to thereference sample, as well as the differences in the X and Y chromosomes.There were no other notable differences in the amount of nucleic acidsbetween the samples.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

1. A method of determining a nucleotide sequence difference betweennucleic acid in a test sample and nucleic acid in a reference sample,comprising: amplifying nucleic acid from a test sample and amplifyingnucleic acid from a reference sample, wherein one of the amplificationreactions is conducted using dUTP and not dTTP and the other isconducted using dTTP and not dUTP; hybridizing to a nucleic acid array asolution comprising the amplified test sample and amplified referencesample; and determining the relative amount of hybridized test andreference nucleic acids bound to the array, wherein a difference in therelative amount of hybridized test and reference nucleic acids bound tothe array identifies the presence of a nucleotide sequence difference.2. The method of claim 1 wherein said amplified test sample is labeledwith a detectable label.
 3. The method of claim 1 wherein said amplifiedreference sample is labeled with a detectable label.
 4. The method ofclaim 1 wherein said amplified test sample and said amplified referencesample are labeled with a detectable label.
 5. The method of claim 4wherein said amplified test and reference samples are labeled with thesame detectable label.
 6. The method of claim 1, wherein said testsample comprises genomic DNA.
 7. The method of claim 1, wherein saidtest sample comprises cDNA.
 8. The method of claim 5, wherein saiddetectable label is a fluorochrome.
 9. The method of claim 2, whereinsaid detectable label is dCTP-Cy3.
 10. The method of claim 2, whereinsaid amplified nucleic acid is label with a detectable label by using anucleotide labeled with a detectable label in the step of amplifyingsaid test sample.
 11. The method of claim 5, wherein said step ofdetermining the relative amount of hybridized test and reference nucleicacids bound to the array comprises: a) determining a signal for thedetectable label hybridized to the array representing the total ofhybridized test and reference nucleic acid; b) treating the hybridizednucleic acids with an enzyme that selectively degrades DNA having uracilresidues; c) determining a signal for the detectable label hybridized tothe array following step b), which signal represents one of saidhybridized test or reference nucleic acid; and d) determining a signalfor the other of the hybridized test or reference by using the signalfrom a) and c).
 12. The method of claim 11, wherein said enzyme thatselectively degrades DNA having uracil residues is uracil-DNAN-glycosylase (UNG).
 13. The method of claim 11, wherein said testsample is obtained from a patient.
 14. The method of claim 11, whereinsaid test sample obtained from a patient is suspected of comprisingcancerous cells.
 15. The method of claim 11, wherein said test sample isobtained from a prenatal specimen.
 16. The method of claim 11, whereinsaid test sample is obtained from an embryo or a fetus.
 17. The methodof claim 11, wherein said test sample is from an individual and themethod is used to detect a genetic abnormality.
 18. The method of claim1, wherein said test sample comprises genomic nucleic acid.
 19. Themethod of claim 1, wherein said test sample comprises cDNA.